Abstract

We have studied the formation of L-leucine nanoparticles under various conditions using an aerosol flow reactor method. Temperatures and L-leucine concentrations for the experiments were selected to vary the saturation conditions for L-leucine in the reactor. In the two extreme cases, L-leucine is either in (i) the condensed phase (110) or completely in (ii) the vapour phase (200) for all concentrations; (iii) at the intermediate temperature (150), the extent of evaporation of L-leucine depends notably on the concentration, and thus partial evaporation and production of residual particles are expected. The size distribution of particles and the particle morphology varied according to formation mechanism with the geometric mean diameter of the particles between 30 nm and 210 nm. Hollow, spherical particles were obtained with the droplet-to-particle method without vaporisation of L-leucine; whereas leafy-looking particles were produced by homogeneous nucleation of supersaturated L-leucine vapour and subsequent growth by heterogeneous vapour deposition.

1. Introduction

The ability to
design and produce pharmaceutical particles with the desired surface properties
by particle engineering or by particle surface modification (e.g., coating)
offers a great advantage in today’s market. Much study has been devoted to the
production of stable and dispersible dry-powder aerosol formulations for
efficient pulmonary delivery of pharmaceutical agents [1–3].

Amino acid
L-leucine is an effective excipient that improves the properties of powder such
as dispersibility, flowability, and stability [2, 4–7]. Surface
accumulation of L-leucine on drug particle surfaces has been achieved by powder
blending [1, 2, 7], surface-diffusion in spray-drying [5, 6] and physical
vapour deposition (PVD) [4, 8]. Among these techniques, the PVD method enables
both efficient tailoring of the characteristics of an L-leucine particles and
preparation of the coating layers on the surface of the core particles that
affects significantly the properties of coated particles [4]. The interaction
between particles can be further reduced with the addition of L-leucine
nanoparticles, that is, glidants adsorbed on the surface of the micron-sized
particles. The glidants decrease the contact area between particles and
increase the separation distance depending on the size and shape of the
glidants [9, 10].

In the gas-phase
methods including spray drying and PVD, the particle size and shape are
affected by the formation mechanism, that is, droplet-to-particle and
gas-to-particle conversion [8, 11]. Under suitable conditions these methods
enable the production of nanoparticles with well-defined structure and size.
The residence times in the heated zone are short preventing the material
decomposition [8]. Dry nanoparticles are collected directly from the gas phase
and are available for use, for example, as glidants without further
purification or processing.

In our previous
study of L-leucine particles, the evaporation of L-leucine took place from the
aerosol L-leucine particles whose mass median particle size was initially
larger than 0.8 m [8]. In work presented in this
paper, we have extended the study to the particle sizes smaller than 0.8 m. The particle number concentrations in the
studies are close with only 13% difference in maximum. It is known that the
thermodynamic phenomena of material such as melting and evaporation may change
remarkably as particle size decreases [12]. Accordingly, a rapid solvent
evaporation from the nanodroplets as well as the small initial size of the dry
particles is likely to enhance the vaporisation of L-leucine due to the Kelvin
effect [12]. As a consequence, the formation of L-leucine nanoparticles in the
later stage changes. We aim to deepen the knowledge of L-leucine evaporation
and particle formation in the gas phase when L-leucine is introduced as
particles notably smaller than in our previous study.

2.2. Nanoparticle Production

Figure 1 shows
the experimental set-up of the reactor. The precursor solution was atomised
with a constant output atomiser in the nonrecirculation mode (model 3076, TSI Inc. Particle Instruments, USA)
in the feeding Section 1 producing droplets around 300 nm estimated by the
manufacturer. A flow rate of 3.5 L/min of carrier gas (nitrogen, )
was used. The average feed rate of precursor solutions through the atomiser was
0.4 mL/min controlled with a needle valve. The actual L-leucine concentration
in the reactor was
0.012, 0.029, 0.065, 0.143, and 0.318 g/ depending on the
L-leucine precursor solution concentrations 1.1, 2.2, 4.3, 8.6, and 17.2 g/L, respectively,
since only fraction of the droplets with suitable size is carried to the heated
zone (Section 2) while the excess liquid is collected to the waste bottle
(Figure 1).

Figure 1: The experimental
of the aerosol flow reactor used in the particle production.

After
atomisation, the droplets were carried to the heated zone (Section 2) of the
reactor, which consisted of a stainless steel tube (inner diameter of 30 mm and
length of 1200 mm) placed inside the furnace. The flow in the heated zone was
fully laminar. Experiments were carried out at three temperatures 110, 150, and
200; and the average residence times in the heated part were 11.1,
10.1, and 9.0 s, respectively. The average residence time (second)
was
calculated using the temperature corrected volume flow rate () the
cross-sectional area of the tube () and the length of the heated zone . The centreline
gas temperatures of the reactor tube were measured with the gas flow at 5 cm intervals with a
-type thermocouple. These temperatures were used in the calculations described
in Section 2.4.1.

The aerosol
exiting the heated zone was rapidly cooled with a large volume of at ambient temperature at a flow rate of 30 L/min using a porous tube (sintered
metal tube with pore size 20 m). The length of the porous tube was 200 mm with
inner diameter of 12.7 mm. In Section 3, the nucleation and subsequent
condensation of L-leucine vapour was initiated. The simultaneous cooling and
dilution with a dilution ratio of 9.6:1 prevented solvent condensation and
decreased the wall deposition losses of dry L-leucine particles. Complete
mixing of aerosol and dilution gas before particle sampling was ensured with a
mixing tube with an inner diameter of 9.6 mm and length of 250 mm. The flow
Reynolds number in the diluter and the mixing tube indicated
turbulent flow.

2.3. Instrumentation and Characterisation

The particle
number size distributions were measured with a differential mobility analyser,
DMA (model 3081, TSI Inc. Particle Instruments, USA) connected to a condensation nucleus counter, CPC (model 3022, TSI Inc. Particle Instruments, USA).
Particle samples were collected with a point-to-plate electrostatic
precipitator, ESP (InTox Products, USA) on carbon-coated copper grids. The morphology of the particles
was studied with a transmission electron microscope, TEM (Philips CM-200, FEG/STEM, FEI Company, The Netherlands)
and a field-emission low-voltage scanning electron microscope, FE-SEM (Leo Gemini DSM982, Leo Electron Microscopy Inc., Germany). The
crystallinity of the individual particles was analysed from the electron
diffraction patterns by TEM. For the SEM imaging, the samples were coated with
platinum to increase the stability of the particles in the electron beam. The
chemical identity of the L-leucine powder before and after the experiments was
studied with - and -NMR (200 MHz Varian Gemini 2000, Varian Inc. Corporate, USA) using as the solvent. The NMR studies confirmed that the chemical composition of
L-leucine did not change during the particle production.

2.4. Reactor Conditions

2.4.1. Saturation Ratio of L-Leucine in the Reactor

The vapour
pressure and saturation ratio of L-leucine in the reactor were calculated using
the enthalpy of sublimation (kJ/mol at K)
according to Svec and Clyde [13]; and it is
described in detail in the article of Raula et al. [8]. The vapour pressure and
saturation ratio of water in the reactor were calculated using same method and
using the saturation vapour pressure of water given in the literature [14]. The
saturation ratios of water remained well below zero for all
reactor conditions including the cooling section of the reactor.

2.4.2. Vaporisation of L-Leucine from the DryParticles in
the Heated Zone

The evaporation time of L-leucine from
the dry particles (i.e. particles contain no water) in the heated zone of the
reactor was estimated using a diffusional transport equation according to
Flagan and Seinfeld [15]: where is the particle diameter, is time, is the diffusion
coefficient of the L-leucine vapour, M is the molecular weight of L-leucine, R is the gas constant, is the particle density, T is the reactor temperature, is the L-leucine vapour
pressure in the environment far away from the particle, is the vapour pressure over the particle surface,
and is the
Fuchs-Sutugin interpolation factor for bridging the equations for continuum and
the free molecular regions during the particle size change due to evaporation
of L-leucine as described by Fuchs and Sutugin [16]. is calculated based on the transfer of L-leucine to
the gas phase by vaporisation of the particle and it is assumed to be zero at
the beginning of calculations. The size and number concentration (N) of
L-leucine particles produced at 110 were used as the input in the calculations.
The particles were assumed to be spheres with monodisperse size distribution
. In (1), the particle diameter is the mass median diameter .
Accordingly, the geometric number mean diameters were converted using a
Hatch-Choate conversion equation (HC) [17] into mass median diameters (see
Table 1) that were used as the initial particle diameters . This gives slight overestimate of the mass median
diameter. The number of particles was the average of the particle
concentrations and it was used together with to calculate iteratively . The particle density was assumed to be the same as
the density of crystalline L-leucine (1.293 g/) given in
literature [18]. No wall losses were taken in to account in the calculations. The
amount of L-leucine vapour in the heated zone increases as the particle size
decreases with time in the calculations as long as the saturation vapour
pressure is reached. Instantaneous and perfect mixing is assumed in the
calculations.

Table 1: Characteristics of L-leucine
nanoparticles produced using different precursor solution concentrations and at
different temperatures in the reactor heated zone.

Fuchs-Sutugin interpolation factor is calculated as where is a Knudsen number for the particle and is calculated iteratively for the
decreasing particle size with the following equation: The diffusion coefficient of the vapour
was estimated using the equation derived from literature [17]: where n is the number of vapour molecules taken to be the same as the number of gas
molecules and is the collision diameter of the molecule that is
considered to be the same as the diameter of the molecule that is derived
from the literature [19].

The Kelvin effect expresses the increase
in vapour pres- sure over a curved surface. The vapour pressure over the particle
surface is the
product of the vapour pressure over the flat surface : where is
surface tension or surface free energy. Since there are no data available on
the surface free energy of L-leucine, the surface tension of the 17.2 g/L aqueous
solution of L-leucine was used instead. This concentration is very close to the
solubility limit of L-leucine in ion-exchanged water. The measured surface
tension of the L-leucine solution (67 mN/m) agrees well with the literature
values of Gliński et al. [20]. A similar assumption is also made in studies of
Lechuga-Ballesteros and Kuo [6].

3. Results

3.1. Reactor Conditions

Figure 2 shows
the vapour pressures of L-leucine in the reactor at concentrations between
0.012 and 0.32 g/ as well as the equilibrium vapour pressure of
L-leucine in the temperature range 120 and 200 calculated using equations and
methods introduced by Raula et al. [8]. Accordingly, the evaporation of
L-leucine begins when the temperature is raised above 140; and L-leucine is
completely vaporised in all concentrations when the temperature is above 180
within the studied concentration range. An infinite residence time is assumed
in these calculations. Table 1 lists the saturation ratios for the different concentrations of L-leucine assuming that all L-leucine is
vaporised. indicates how far the condition in
the heated zone is from the actual L-leucine vaporisation.

In the cooling
zone, the cooling rates of the aerosol were 210 and 293/s from 150 and 200
to ambient temperature . The
rapid cooling induced a sudden change in saturation conditions leading to the
supersaturation of L-leucine vapour. This resulted in saturation ratios
temporarily far above unity (from 24.8 to 310) based on calculations of
saturation ratios using equations and methods introduced in Raula et al. [8].
Instantaneous perfect mixing was assumed; and the possible temperature gradient
between the centre line and the walls was not taken into account.

3.2. Vaporisation of L-Leucine from the Dry Particles in
the Heated Zone

Figures 3(a) and
3(b) show the change in size of the particles due to the evaporation of
L-leucine at 150 and 200. In general, the time needed for the particle size
to reach an equilibrium state was short compared to the residence time in the
heated zone of the reactor. At 200, the time needed for complete evaporation
ranged from 11 to 38 milliseconds for particle sizes of 418 and 782 nm,
respectively (see Figure 3(b)). The reduction rate of the particle size decreased
from 38 to 20 nm/ms as the initial particle size increased from 418 to 782 nm.

Figure 3: Calculated size
reduction of L-leucine particles with different initial particle size as a
function of time at (a) 150 and (b) 200.

Based on the
estimated saturation ratio (0.48) for L-leucine at 150 calculated according to Raula et al. [8]
L-leucine was expected to vaporise completely at the lowest concentration ( of 0.012 g/). However the calculations with the diffusional transport equation (1) showed only partial vaporisation producing residual
particles of 260 nm, see Table 2. The time required to reach equilibrium size
was notably longer than at 200 varying from 3 to 0.8 seconds for particle
sizes of 418 and 782 nm, respectively. According to the calculations, complete
evaporation of L-leucine with initial particle size of 418 nm corresponding to
a concentration of 0.012 g/ took place at 157 in 606 milliseconds.

Table 2: The comparison of the vaporisation
of L-leucine from the particles in the heated zone of the reactor in the
current and previous study of Raula et al. [8]. Experimentally derived particle
number concentrations in the calculations were in this study
and in Raula et al. [8].

3.3. Number Size Distributions

Table 1 shows
the characteristics of the particles produced at different reactor temperatures
and L-leucine concentrations .
The size distributions of particles produced at 110 are shown in Figure 4. At
110, the solute droplets were merely dried. The geometric number mean
diameter of the
particles increased from 99 to 150 nm with increasing L-leucine concentration
(Table 1). The GSDs were around 2 at every concentration. The number
concentration of particles (N) varied
only slightly between the experiments and was on average L/.
The size distributions were unimodal and close to symmetrical, that is, was very close to the
count median diameter (CMD) obtained in the measurements with only a few
nanometres difference in all cases. Therefore, the corresponding mass mean
diameters of the size distribution (see Table 1) were calculated with
Hatch-Choate equation described by Hinds [17].

Figure 4: Number size
distributions of L-leucine particles produced from the reactor at 110.
L-leucine concentration varied from 0.012 to 0.32 g/.

Figure 5 shows
the number size distributions of the L-leucine particles produced at 150.
According to the calculated saturation ratio, L-leucine was completely
vaporised only with the lowest concentration of L-leucine, that is 0.012 g/,
while at higher concentrations than that, L-leucine was partially vaporised. of the particles increased
from 31 to 209 nm with increasing L-leucine concentration and the GSD varied
between 1.4 and 1.8. N did not change
notably with respect to the L-leucine concentration and was between and L/. The size distributions were close to
log-normal except for the highest concentration where a bimodal distribution
was obtained.

Figure 5: Number size
distributions of L-leucine particles produced from the reactor at 150.
L-leucine concentration varied from 0.012 to 0.32 g/.

Figure 6 shows the number size
distributions of the particles from the reactor at 200, where L-leucine was
completely vaporised at all concentrations according to S (see Figure 2). of the particles increased from 28 to 162 nm and N increased from to L/ with increasing L-leucine concentration. With the two lowest L-leucine
concentrations, the size distributions were unimodal. With of 0.065 g/ and above, bimodality
in the distributions was observed.

Figure 6: Number size
distributions of L-leucine particles produced from the reactor at 200.
L-leucine concentration varied from 0.012 to 0.32 g/.

3.4. Shape and Structure of L-Leucine Nanoparticles

Figures 7(a) and 8(a) show SEM images of
L-leucine nanoparticles produced with the highest concentration of L-leucine
(0.32 g/) from the reactor at 110 and 150, respectively. The
sizes of the particles are in a good agreement with
volume-based median
diameters calculated by Hatch-Choate conversion equations [17]: the
diameters ranged from 418 to 782 nm. In both cases, the particles appeared
collapsed and partially fractured indicating that particles are produced by
droplet drying. TEM images (Figures 7(b) and 8(b)) show relatively low contrast
in the central areas of the particles so that the supporting film underneath
can be clearly seen. For this to happen with this size of particles, the
particle shell has to be very thin which indicates low particle density. Figures
9(a) and 9(b) show the SEM and TEM images, respectively, of L-leucine
nanoparticles produced at the highest concentration (0.32 g/) with
the temperature of the heated zone at 200. At this temperature L-leucine
vaporises and the particles were formed by the nucleation and condensation of
the L-leucine vapour. The particles had a light, leafy-looking structure that
is clearly different from the particle structures obtained at lower
temperatures.

Electron diffraction patterns taken from
a population of particles in TEM are shown as inserts in the corresponding TEM
images (Figures 6–8(b)). The
diffraction patterns indicate that the particles are crystalline despite the
weak contrast due to the deterioration of particles within a few seconds under
the electron beam.

4. Discussion

Table 2 shows
the amount of L-leucine fed to the reactor and the changes in particle size due
to the vaporisation of L-leucine calculated in this study and compared with the
values of the previous study of Raula et al. [8]. Even though the particle
number concentrations are of the same order of magnitude with only 13% difference,
the initial droplet size in the experiments is considerably different.
According to the manufacturer (TSI), the droplets around 300 nm are produced
with the atomizer used in this study whereas the ultrasonic atomizer used by
Raula et al. [8] generates micron-sized droplets. This in turn affects the dry
particle size as well as the amount of L-leucine vapour in the heated zone of
the reactor.

According to the
calculations at 150, no vaporisation of L-leucine was observed with particles
larger than 0.75 m, that is, in the study of Raula et al. [8] and the largest
particle size in this study. Below 0.75 m, the calculations showed the partial
vaporisation of L-leucine and formation of the residual particles with the mass
median diameters 0.26, 0.44, 0.50, and 0.54 m (Table 2). The partial vaporisation observed even
with the smallest particle size corresponding to the lowest concentration is in
contradiction with the saturation ratio that predicted complete vaporisation
(Table 1). Since the amount of L-leucine in the reactor was derived from the
total number of particles, particle size, and density as described in Section 2.4.2, the calculation is
likely to give a slight overestimation of the amount of L-leucine in the
reactor. One reason for this is the density value of crystalline bulk L-leucine
used in the calculations compared to the hollow particle structure observed in
TEM images. The repetition of the calculations at temperature of 157 showed
full vaporisation of L-leucine at the lowest concentration (0.012 g/).
This indicates that saturated conditions are close and only small variations
may affect particle formation in these conditions.

A complete
vaporisation of L-leucine was observed at 200 for particles
smaller than 1.69 m. The time needed for the vaporisation
increased exponentially with increasing particle size as the mass of L-leucine
increased. The vaporisation of L-leucine decreases the particle size and it
further accelerates the rate of vaporisation of the particles (see Figure 3(b)).
This is understood by the Kelvin effect, which means that thermal transitions
like melting and vaporisation of material requires less energy when particle
size decreases [21–24]. For particles composed of organic material the
Kelvin effect can be significant for particle sizes up to 200 nm [12].

4.2. Formation of Dry L-Leucine Particles

At 110, the particles were formed by
droplet drying and the cooling of the aerosol did not affect the formation of
the particles or the resulting particle size and size distribution. During the
solvent evaporation, L-leucine accumulated on an air-water interface
that, in this study, is the surface of the droplets. L-leucine is also known to
spontaneously assemble as structured aggregates at the interfaces of the
aqueous solution as described by Weissbuch et al. [25]. The rapid formation of
the L-leucine surface layer caused the pressure build-up by the vaporisation of
water expanding the surface layer. This resulted hollow, collapsed, and
fractured particles with low density as observed also in the study of Raula et al. [8]. The volume-based median diameters of the particles coincide well with
the observed particles.

The layer formation notably affected the
size of L-leucine particles produced with a droplet-to-particle method at 110. Let us assume that L-leucine particles are spherical with the density of
crystalline L-leucine (1.293 g/). According to the manufacturer,
the atomizer generates water droplets with diameter around 300 nm. Based on
these data, the sizes of the solid, spherical L-leucine particles for every
solution concentration can be calculated. The diameter of the particles at a
concentration of 0.012 g/ was 3.6 times larger than the calculated
size. The increase in the particle size was evidently caused by the expansion
of the particle surface layer as discussed above. The size difference decreased
with increasing L-leucine concentration, but was still 2.1 at the highest
L-leucine concentration, 0.32 g/. This indicates a very low
density of the particles. The micron-sized porous particles with the density as low as 0.4 g/ has
been previously prepared by emulsification and solvent evaporation by Edwards
et al. [26].

At 200, L-leucine fully evaporated in
the reactor at all concentrations. High supersaturation and subsequent
homogeneous nucleation of the L-leucine vapour were obtained in the cooling
zone. During the vapour deposition on the surface of the nuclei, L-leucine
formed leafy-looking crystallites with sizes of a few nanometres with similar
morphology of particles than observed by Raula et al. [8]. The heterogeneous
deposition of L-leucine vapour is preferred on discontinuous spots such as
edges and corners if present as studied by Rogers and Yau [27]. Apparently, the
discontinuous domains for heterogeneous nucleation were created at the very
beginning of the formation of stable nuclei by homogeneous nucleation of vapour
resulting in crystal growth in a certain direction (see Figure 9). The number
size distribution of the resulting particles was relatively narrow and unimodal
at low L-leucine concentrations. The particle size was more dependent on the
L-leucine concentration and it varied from 28 nm up to 162 nm compared to the
particles prepared by Raula et al. [8] at 200 with geometric mean diameters close
to 100 nm in all cases except at the lowest concentration. Increasing the
vapour concentration resulted in the appearance of shoulders in the
distribution. This indicated that several nucleation modes were occurring along
the cooling of the L-leucine vapour. There could be several reasons for that
such as the trace amounts of unvaporised impurities in the precursor solution.
Another reason is the temperature gradient in the cooling zone that was caused
by the introduction of ambient nitrogen through the porous tube, that is, through
the tube walls. The bimodal size distribution obtained at the high supersaturations of L-leucine vapour was due to the nonuniform temperature field and turbulent flow profile in the cooling and mixing zone as well as due to the slow diffusion of L-leucine vapour.

At the intermediate temperature of 150,
the extent of evaporation of L-leucine depended on the concentration as
discussed in Sections 3.2 and 4.1. The saturated conditions are close and only
small variations may affect particle formation. In fact, the size of the
particles produced at the lowest concentration agreed well with the size obtained at 200
with the same concentration and strongly indicated particle formation purely by
vapour nucleation. The reactor conditions for L-leucine became saturated above
a concentration of 0.037 g/ corresponding to 2.0 g/L, at which the
saturation ratio was unity. A fraction of the vaporised L-leucine (0.037 g/)
was deposited on the surface of the residual, unvaporised solid L-leucine
particles via heterogeneous vapour deposition. The particle size was clearly
dependent on both the L-leucine concentration and on the vapour deposition of
the L-leucine vapour on the residual particles. Narrow-size distributions were the
result of the effective heterogeneous vapour deposition of L-leucine. In
heterogeneous condensation/vapour deposition, which is the reverse of size
decrease by evaporation described in (1), the smaller particles grow faster
compared to larger particles narrowing the size distribution as described by
Hinds [17]. However, the small amount of L-leucine vapour deposited on the
particle surfaces was not a major factor determining the particle size at high
L-leucine concentrations (0.14 and 0.32 g/). These concentrations
yielded particle sizes larger than those produced at 110 as summarised in
Table 1. This difference in size can be explained by the formation of the
L-leucine layer on the droplet surface that increased vapour pressure of the
solvent water inside the droplet with the elevated temperature in the heated
zone. This led to further expansion of the L-leucine surface layer during
drying. The second mode as a shoulder in the size distribution at 0.32 g/ in the reactor at 150 may denote the appearance of fractured particles as was
observed also in SEM images (Figure 8).

5. Conclusions

The production
and formation of L-leucine nanoparticles under various conditions have been studied
using the aerosol flow reactor method. The particle formation and growth
mechanisms depended on the L-leucine saturation conditions in the reactor. At
110, low-density particles were formed by droplet drying. The particle size
was determined solely by the precursor solution concentration with between 99 and 150 nm. At
15, L-leucine was partially vaporised and some unvaporised material remained
in residual particles. The particle size increased from 31 nm up to 209 nm. The
particle size depended not only on the precursor solution concentration but
also on the heterogeneous condensation of L-leucine vapour on the residual
particles. At 200, L-leucine was entirely vaporised at all precursor
concentrations and the particle size varied from 30 nm to 210 nm. The
vaporisation of L-leucine was faster compared to the previous studies.
Furthermore, the particle size was more dependent on the amount of L-leucine in
the reactor. The particles were formed by homogeneous nucleation of L-leucine
vapour causing the formation of leaf-like, crystalline nanoparticles as also
observed previously.

Acknowledgments

The authors would like to thank Academy of Finland for financial support. The
authors also wish
to thank Doctor Hua Jiang for work with the transmission electron microscope
and Doctor Jouni Pyykönen for valuable help in calculations.